1. Field of the Invention
The present invention relates to a process for the production of a human growth hormone, more specifically to a human growth hormone secretion plasmid which allows the secretion and accumulation of a human growth hormone having a molecular weight of 20,000 (hereinafter referred to as 20 kD hGH) into the periplasm of Escherichia coli, and to a process for the production of the human growth hormone using a transformed strain of E. coli in which the said plasmid is introduced.
2. Description of the Prior Art
A human growth hormone having a molecular weight of 22,000 (hereinafter referred to as 22 kD hGH) is today used for treatment of pituitary dwarfism. Furthermore, use of the human growth hormone is expected to be expanded to, for example, the treatment of disorders such as chronic renal insufficiency in childhood, bone fractures and burns. However, 22 kD hGH is known to have side effects. As to the side effects of 22 kD hGH, it has been pointed out from the results of animal experiments that 22 kD hGH may cause leukemia when used for treatment of human patients (Rogers et al., The Lancet, 434, Aug., 27, 1977). In fact, up to 1987, the incidence of leukemia in populations who use the human growth hormone increased ten times more than anticipated, which cannot be ignored by any means (Fisher et al., The Lancet, 1159, May 21, 1988; Watanabe et al., The Lancet, 1159, May 21, 1988). As to diabetogenic activity, which is another side effect caused by 22 kD hGH, it was reported that 30% of patients who received 22 kD hGH treatment in clinical trial in Turner's syndrome showed a decrease in glucose tolerance (HORUMON TO RINSHO (Hormone and Clinical medicine), 38, 155-159, 1990). In order to develop new applications for the human growth hormone, it is strongly desired to make the human growth hormone less likely to induce side effects for practical use in the clinical field.
Besides the 22 KD hGh which is presently used, another known human growth hormone is 20 kD hGH. This 20 kD hGH is reported to have lower growth stimulating activity in a lymphoma cell line than 22 kD hGH (Endocrinol. Japon, 36(1), 9-13, 1989) and have no or reduced diabetogenic activity (Endocrinol. Japon, 34, 73-85, 1987).
Recently, it has become possible to produce the human growth hormone intracellularly, extracellularly or in the periplasm by means of recombinant DNA technology in which a gene of human growth hormone is expressed in a microorganism as a host. As to the extracellular production of 22 kD hGH, a process for the secretion and production of 22 kD hGH using Bacillus subtilis (Japanese Patent Laid-open No. 273591/1989) has been reported. In a 20 kD hGH production using this process for the extracellular production of 22 kD hGH, a large portion of the expressed product was intracellularlly accumulated in a precursor form that contained secretion signal of neutral protease of Bacillus amyloliquefaciens preceding 20 kD hGH, and is therefore different from the authentic 20 kD hGH. In this case, a small portion of 20 kD hGH expressed has been successfully secreted and accumulated at a concentration of about 1 mg/1.multidot.A660 in a culture, as a result of intensive studies on culture methods. However, the concentration of 20 kD hGH thus accumulated is not considered to be sufficient from the viewpoint of industrial production, and thus further studies are needed to increase the accumulation of 20 kD hGH. However, in this case, it is impossible to subject the culture supernatant directly to column chromatography using ordinary methods, since the medium used contained salts and polypeptides in high concentration. Thus, culture supernatant must be diluted 5-10 times prior to the column chromatography. This operation requires a big column chromatography apparatus and is disadvantageous in terms of production cost. Thus, as to the extracellular production of 20 kD hGH on an industrial scale, any satisfactory method has not yet been established.
On the other hand, as to the intracellular production of human growth hormone, Miyamoto et al. extensively studied the intracellular production of 22 kD hGH and 20 kD hGH using E. coli (Miyamoto et al., International Symposium on GRF, Growth Hormone and Somatomedin Program and Abstracts, 28, Nov. 1-2, 1986). With the aim of attaining the highly efficient expression of 20 kD hGH, Miyamoto et al. tried to use a method which had already been established for 22 kD hGH production to attain this highly efficient expression of 20 kD hGH. However, it was reported that the amount of 20 kD hGH expressed was only 1/10 that of 22 kD hGH. These facts suggest that 20 kD hGH cannot be effectively expressed intracellularly in E. coli by simply applying methods which has already been established for production of 22 kD hGH, to the 20 kD hGH production and that there are problems unique to the expression of 20 kD hGH. Namely, any satisfactory method for production of 20 kD hGH on an industrial scale has not yet been established even by intracellular production using E. coli, which is technically easier than production using secretion process.
As to secretion into the periplasm of E. coli, Gray et al. reported the secretion of 22 kD hGH (Gray et al., Gene, 39, 247-254, 1985). The periplasm refers to a space between the inner membrane and the outer membrane of gram-negative bacteria including E. coli. In gram-negative bacteria, a precursor protein containing a signal sequence is proceeded during secretion to cleave the secretion signal sequence that is necessary for membrane penetration upon passing through the inner membrane. The protein, which now becomes the matured protein by losing its secretion signal, generally cannot pass through the outer membrane and is accumulated in the periplasm. For secretion and accumulation of 22 kD hGH in the periplasm of E. coli, Gray et al. constructed a plasmid in which the 22 kD hGH gene is correctly ligated to 3'-terminal end of the region for secretion signal of E. coli alkaline phosphatase gene (phoA), and introduced this plasmid into E. coli. This report of Gray et al relates to 22 kD hGH and contains no description as to 20 kD hGH.